A trehalose biosynthetic enzyme doubles as an osmotic stress sensor to regulate bacterial morphogenesis.

The dissacharide trehalose is an important intracellular osmoprotectant and the OtsA/B pathway is the principal pathway for trehalose biosynthesis in a wide range of bacterial species. Scaffolding proteins and other cytoskeletal elements play an essential role in morphogenetic processes in bacteria. Here we describe how OtsA, in addition to its role in trehalose biosynthesis, functions as an osmotic stress sensor to regulate cell morphology in Arthrobacter strain A3. In response to osmotic stress, this and other Arthrobacter species undergo a transition from bacillary to myceloid growth. An otsA null mutant exhibits constitutive myceloid growth. Osmotic stress leads to a depletion of trehalose-6-phosphate, the product of the OtsA enzyme, and experimental depletion of this metabolite also leads to constitutive myceloid growth independent of OtsA function. In vitro analyses indicate that OtsA can self-assemble into protein networks, promoted by trehalose-6-phosphate, a property that is not shared by the equivalent enzyme from E. coli, despite the latter's enzymatic activity when expressed in Arthrobacter. This, and the localization of the protein in non-stressed cells at the mid-cell and poles, indicates that OtsA from Arthrobacter likely functions as a cytoskeletal element regulating cell morphology. Recruiting a biosynthetic enzyme for this morphogenetic function represents an intriguing adaptation in bacteria that can survive in extreme environments.

Detection of biogenic amines in C57BL/6 mice brain by capillary electrophoresis electrokinetic supercharging.

Ischemic stroke is caused when blood flow to the brain is stopped and is a major cause of death and long term disability across the globe. Excessive release of neurotransmitters is triggered in the brain by ischemia that mediates neuronal damage and causes ischemic injury. In this study, a simple, sensitive, and on-line preconcentration capillary electrophoresis method based on electrokinetic supercharging (EKS) was developed for the determination of the biogenic amines including dopamine (DA), epinephrine (E), and norepinephrine (NE) in C57BL/6 mice brain. Under the optimized conditions, the analytes were concentrated and detected within 10 min. The detection limits for the analytes ranged from 0.42 to 0.57 ng mL(-1) for a mice brain matrix. With the proposed method, the analyses of three neurochemical amines in C57BL/6 mice brain tissue during cerebral ischemic/reperfusion had been performed successfully.

Calcium plays a key role in paraoxon-induced apoptosis in EL4 cells by regulating both endoplasmic reticulum- and mitochondria-associated pathways.

CONTEXT AND OBJECTIVE: Paraoxon (POX) is one of the most toxic organophosphorus pesticides, but its toxic mechanisms associated with apoptosis remain unclear. The aim of this study was to investigate calcium-associated mechanisms in POX-induced apoptosis in EL4 cells. MATERIALS AND METHODS: EL4 cells were exposed to POX for 0-16 h. EGTA was used to chelate Ca(2+ ) in extracellular medium, and heparin and procaine were used to inhibit Ca(2+ )efflux from the endoplasmic reticulum (ER). Z-ATAD-FMK was used to inhibit caspase-12 activity. The apoptotic rate assay, western blotting and immunocytochemistry (ICC) were used to reveal the mechanisms of POX-induced apoptosis. RESULTS AND DISCUSSION: POX significantly increased the expression and activation of caspase-12 and caspase-3, enhanced expression of calpain 1 and calpain 2, and induced the release of cyt c, but did not change the expression of Grp 78. Inhibiting caspase-12 activity alleviated POX-induced upregulation of calpain 1 and caspase-3, promoted POX-induced upregulation of calpain 2, and reduced POX-induced cyt c release, suggesting that there was a cross-talk between the ER-associated pathway and mitochondria-associated apoptotic signals. Attenuating intracellular calcium concentration with EGTA, heparin or procaine decreased POX-induced upregulation of calpain 1, calpain 2, caspase-12 and caspase-3, and reduced POX-induced cyt c release. After pretreatment with EGTA or procaine, POX significantly promoted expression of Grp 78. CONCLUSIONS: Calcium played a key role in POX-induced apoptosis in EL4 cells by regulating both ER- and mitochondria-associated pathways. The cross-talk of ER- and mitochondria-associated pathways was accomplished through calcium signal.

Protein quality control and aggregation in the endoplasmic reticulum: From basic to bedside.

Endoplasmic reticulum (ER) is the largest membrane-bound compartment in all cells and functions as a key regulator in protein biosynthesis, lipid metabolism, and calcium balance. Mammalian endoplasmic reticulum has evolved with an orchestrated protein quality control system to handle defective proteins and ensure endoplasmic reticulum homeostasis. Nevertheless, the accumulation and aggregation of misfolded proteins in the endoplasmic reticulum may occur during pathological conditions. The inability of endoplasmic reticulum quality control system to clear faulty proteins and aggregates from the endoplasmic reticulum results in the development of many human disorders. The efforts to comprehensively understand endoplasmic reticulum quality control network and protein aggregation will benefit the diagnostics and therapeutics of endoplasmic reticulum storage diseases. Herein, we overview recent advances in mammalian endoplasmic reticulum protein quality control system, describe protein phase transition model, and summarize the approaches to monitor protein aggregation. Moreover, we discuss the therapeutic applications of enhancing endoplasmic reticulum protein quality control pathways in endoplasmic reticulum storage diseases.

Differential Regulation of Microglial Activation in Response to Different Degree of Ischemia.

Microglia are primary immune cells within the brain and are rapidly activated after cerebral ischemia. The degree of microglial activation is closely associated with the severity of ischemia. However, it remains largely unclear how microglial activation is differentially regulated in response to a different degree of ischemia. In this study, we used a bilateral common carotid artery ligation (BCAL) model and induced different degrees of ischemia by varying the duration of ligation to investigate the microglial response in CX3CR1GFP/+ mice. Confocal microscopy, immunofluorescence staining, RNA sequencing, and qRT-PCR were used to evaluate the de-ramification, proliferation, and differential gene expression associated with microglial activation. Our results showed that 30 min of ischemia induced rapid de-ramification of microglia but did not have significant influence on the microglial density. In contrast, 60 min of ischemia led to a significant decrease in microglial density and more pronounced de-ramification of microglial processes. Importantly, 30 min of ischemia did not induce proliferation of microglia, but 60 min of ischemia led to a marked increase in the density of proliferative microglia. Further analysis utilized transcriptome sequencing showed that microglial activation is differentially regulated in response to different degrees of ischemia. A total of 1,097 genes were differentially regulated after 60 min of ischemia, but only 68 genes were differentially regulated after 30 min of ischemia. Pathway enrichment analysis showed that apoptosis, cell mitosis, immune receptor activity and inflammatory-related pathways were highly regulated after 60 min of ischemia compared to 30 min of ischemia. Multiple microglia-related genes such as Cxcl10, Tlr7, Cd86, Tnfrsf1a, Nfkbia, Tgfb1, Ccl2 and Il-6, were upregulated with prolonged ischemia. Pharmacological inhibition of CSF1 receptor demonstrated that CSF1R signaling pathway contributed to microglial proliferation. Together, these results suggest that the proliferation of microglia is gated by the duration of ischemia and microglia were differentially activated in responding to different degrees of ischemia.

MouseVenue3D: A Markerless Three-Dimension Behavioral Tracking System for Matching Two-Photon Brain Imaging in Free-Moving Mice.

Understanding the connection between brain and behavior in animals requires precise monitoring of their behaviors in three-dimensional (3-D) space. However, there is no available three-dimensional behavior capture system that focuses on rodents. Here, we present MouseVenue3D, an automated and low-cost system for the efficient capture of 3-D skeleton trajectories in markerless rodents. We improved the most time-consuming step in 3-D behavior capturing by developing an automatic calibration module. Then, we validated this process in behavior recognition tasks, and showed that 3-D behavioral data achieved higher accuracy than 2-D data. Subsequently, MouseVenue3D was combined with fast high-resolution miniature two-photon microscopy for synchronous neural recording and behavioral tracking in the freely-moving mouse. Finally, we successfully decoded spontaneous neuronal activity from the 3-D behavior of mice. Our findings reveal that subtle, spontaneous behavior modules are strongly correlated with spontaneous neuronal activity patterns.

The expression of αA- and ßB1-crystallin during normal development and regeneration, and proteomic analysis for the regenerating lens in Xenopus laevis.

PURPOSE: To explore the expression of the lens crystallins (αA- and ßB1-crystallin) in Xenopus laevis embryonic lens development and regeneration and to analyze the order of different crystallins generated in the regenerating lens. METHODS: Real Time-PCR, Immunofluorescence, and 2D-PAGE were used to analyze the expressions of αA-crystallin and ßB1-crystallin, and related factors during embryonic lens development and regeneration in Xenopus laevis. RESULTS: αA-crystallin and ßB1-crystallin were first detected at stage 29/30 during normal development, and the two crystallins were simultaneously detected in regeneration. During embryonic lens development, the relative expression level of the ßB1-crystallin gene was higher than that of the αA-crystallin gene. In the process of the lens regeneration, however, the relative expression level of the ßB1-crystallin gene was lower than that of the αA-crystallin gene. Throughout embryonic lens development, the two crystallin transcripts showed the same variation trends, and similar occurrence did in the regeneration process. Crystallins showed different localization and distribution during the ontogeny and regeneration, especially in the lens fiber region. 2D-electrophores revealed the patterns of the sequential synthesis of crystallins, with regard to the different classes and apparent variations of some auxiliary regulatory factors. CONCLUSIONS: The ontogeny and localization of the crystallins during embryonic lens development and regeneration indicated a different development program, although they have identical origins, the ectoderm. The expression level of crystallin transcripts displayed a consistent variation tendency, but the presence of appreciable differences was still exposed. In addition to stably producing the crystallins of different classes in accordance with established procedure, these auxiliary factors may perform the function, to some extent, because of significant changes in their expression throughout the process of lens regeneration.

Interaction between Pax6 and its novel mutant in Bufo raddei Strauch.

PURPOSE: Exploration of the relationship between a novel paired box 6 (Pax6) mutant and Pax6 in Bufo raddei Strauch. METHODS: RT-PCR, yeast 2-hybrid system, and co-immunoprecipitation were used to analyze the Pax6 protein and its mutant during embryonic eye development in Bufo raddei Strauch. RESULTS: We have cloned the Pax6 ORF sequence from Bufo raddei Strauch. Here we report the cloning of a novel Pax6 homolog of Bufo raddei Strauch named Pax6 variant. Comparing the 2 genes, the homolog of ORF nucleotide sequence is more than 99% in Bufo raddei Strauch; only the proline-serine-threonine(PST)-rich transaction domain differs. The deduced amino acid sequences of PST region are 53.1% identical. An interaction was found between Pax6 and Pax6 variant via yeast 2-hybrid system; with further study, we found that they interacted in vivo via co-immunopricipitation. CONCLUSIONS: A Pax6 mutant was first found in Bufo raddei Strauch. Interaction between Pax6 and Pax6 variant may play a critical role during eye development in Bufo raddei Strauch. This suggests that expression of Pax6 variant may play a role and appears to be a necessity in eye development, but that Pax6 itself is still pivotal in eye development.

Pharmacological Targeting of CSF1R Inhibits Microglial Proliferation and Aggravates the Progression of Cerebral Ischemic Pathology.

Ischemic stroke can induce rapid activation of the microglia. It has been reported that the microglia's survival is dependent on colony-stimulating factor 1 receptor (CSF1R) signaling and that pharmacological inhibition of CSF1R leads to morphological changes in the microglia in the healthy brain. However, the impact of CSF1R inhibition on neuronal structures and motor ability after ischemia-reperfusion remains unclear. In this study, we investigated microglial de-ramification, proliferation, and activation after inhibition of CSF1R by a tyrosine kinase inhibitor (ki20227) in a mouse model of global cerebral ischemia induced by bilateral common carotid artery ligation (BCAL). In addition to microglial morphology, we evaluated the mRNA expression of cytokines, chemokines, and inflammatory receptors. Our results show that pharmacological inhibition of CSF1R in ischemic mice resulted in the blockade of microglial proliferation and a shift in microglial morphology reflected by excessive de-ramification and a more activated phenotype accompanied by an enhanced innate immune response. Furthermore, we show that pharmacological inhibition of CSF1R in ischemic mice resulted in the aggravation of neuronal degeneration and behavioral impairment. Intravital two-photon imaging revealed that although pharmacological inhibition of CSF1R did not affect the recovery of dendritic structures, it caused a significant increase in spine elimination during reperfusion in ischemic mice. These findings suggest that pharmacological inhibition of CSF1R induces a blockade of microglial proliferation and causes acute activation of the microglia accompanied by a severe inflammatory response. It aggravates neuronal degeneration, loss of dendritic spines, and behavioral deficits after transient global cerebral ischemia.

Increased BBB Permeability Enhances Activation of Microglia and Exacerbates Loss of Dendritic Spines After Transient Global Cerebral Ischemia.

Ischemic stroke can induce rapid disruption of blood-brain barrier (BBB). It has been suggested that increased BBB permeability can affect the pathological progression of ischemic tissue. However, the impact of increased BBB permeability on microglial activation and synaptic structures following reperfusion after ischemia remains unclear. In this study, we investigated microglial activation, dendritic damage and plasticity of dendritic spines after increasing BBB permeability following transient global cerebral ischemia in the somatosensory cortices in mice. Bilateral common carotid artery ligation (BCAL) was used to induce transient global cerebral ischemia. Mannitol was used to increase the BBB permeability. Intravital two-photon imaging was performed to image the dendritic structures and BBB extravasation. Microglial morphology was quantitated using a skeletonization analysis method. To evaluate inflammation of cerebral cortex, the mRNA expression levels of integrin alpha M (CD11b), CD68, chemokine (C-X-C motif) ligand 10 (IP10) and tumor necrosis factor alpha (TNF-α) were measured by fluorescent quantitative PCR. Intravital two-photon imaging revealed that mannitol caused a drastic increase in BBB extravasation during reperfusion after transient global ischemia. Increased BBB permeability induced by mannitol had no significant effect on inflammation and dendritic spines in healthy mice but triggered a marked de-ramification of microglia; importantly, in ischemic animals, mannitol accelerated de-ramification of microglia and aggravated inflammation at 3 h but not at 3 days following reperfusion after ischemia. Although mannitol did not cause significant change in the percentage of blebbed dendrites and did not affect the reversible recovery of the dendritic structures, excessive extravasation was accompanied with significant decrease in spine formation and increase in spine elimination during reperfusion in ischemic mice. These findings suggest that increased BBB permeability induced by mannitol can lead to acute activation of microglia and cause excessive loss of dendritic spines after transient global cerebral ischemia.

In vivo two-photon imaging reveals a role of progesterone in reducing axonal dieback after spinal cord injury in mice.

Progesterone (PG) as a neuroprotective reagent has been used for the treatment of spinal cord injury (SCI) in experimental animal models. However, its effect and mechanism on axonal dieback at the early stage of SCI remain unclear. Here, we investigate the dynamics of injured axons and the effect of PG on the axonal dieback, glial response, and behavioral recovery in a mouse model of SCI. Two-photon intravital imaging combined with a simplified imaging window chamber were used to image axons in hemisected spinal cords over a period of 3 days. Repeated imaging showed that axonal dieback distance in mice treated with PG after SCI was significantly reduced than that in mice treated with vehicle after SCI (P < 0.05) at the time point of 24 h, 48 h, and 72 h after SCI. The densities of astrocytes and microglia in the SCI-vehicle treated group were significantly higher than those in mice treated with PG after SCI (P < 0.05). Real time polymerase chain reaction assay indicated that administration of PG after SCI down-regulated the expression of pro-inflammatory cytokines MCP-1, NOS2, and IL-1ß (P < 0.05). PG treatment also improved the behavioral performance post injury. These findings suggested that PG exerted a neuroprotective effect by attenuating axonal dieback, reducing the accumulation of astrocytes and microglia and inhibiting the release of pro-inflammatory cytokines.

Reversible recovery of neuronal structures depends on the degree of neuronal damage after global cerebral ischemia in mice.

It has been observed by in vivo imaging that damaged neuronal structures can be reversibly restored after ischemic insults with the application of timely therapeutic interventions. However, what degree of neuronal damage can be restored and the time frame for reversible recovery of neuronal structures remain unclear. Here, transcranial two-photon imaging, histological staining and electron microscopy were used to investigate the reversible recovery of neuronal structures from dendrites to soma after different durations of global cerebral ischemia in mice. Intravital imaging revealed that the damage to dendritic structures was reversible when ischemia time was <1h, but they became difficult to restore after >3h of ischemia. Data from fixed YFP brain slice and Golgi staining indicated that the damage of dendritic structures progressively extended to deeper dendritic shafts with the extension of ischemia time. Furthermore, longer duration of ischemia caused an increasing number of degenerating neurons. Importantly, significant chromatin margination and karyopyknosis of neuron were observed after 6h of ischemia. These data suggested that neuronal structures could be reversibly restored when ischemia time was <1h, but irreversible and progressive damage to neurons occurred with longer duration of ischemia. Consistently, behavioral performance of post-ischemic animals experienced an ischemia time-dependent recovery. Taken together, our data suggested that recovery of neuronal structures following ischemia was dependent on the duration of ischemia, and prevention of neuronal loss is a key target for therapeutic interventions in ischemic stroke.

Transient global cerebral ischemia induces rapid and sustained reorganization of synaptic structures.

Ischemia can cause rapid neuronal damage. Previous studies have suggested that synaptic structures and cortical functions can be rescued if therapeutic interventions are applied in time, but the structural basis for this resilience remains incompletely understood. Here, we investigated the restoration of synaptic structures and postischemic plasticity of dendritic spines in the somatosensory cortices of mice by taking advantage of a reversible global cerebral ischemia model. Intravital two-photon imaging revealed that although dendritic structures were rapidly distorted after global ischemia, only a small percentage of spines were actually lost after transient ischemia. Electron microscopy indicated that most presynaptic electron-dense structures were still apposed to postsynaptic densities, and that the majority of disrupted synaptic structures were rapidly reinstated following reperfusion after transient ischemia. Repeated imaging suggested that restored dendrites survived the initial ischemia -reperfusion challenge. Importantly, spines on the restored dendrites underwent a rapid and sustained structural reorganization following transient ischemia. These findings suggested that disrupted synapses during transient ischemia could be rapidly restored after ischemia/reperfusion, and that restored dendritic structures remained plastic to rebuild the cortical network.

Transcriptomic analysis reveals differential activation of microglial genes after ischemic stroke in mice.

Microglia are immune cells in the brain and play a pivotal role in the progression of ischemic injury, but the gene expression and signaling pathways related to the activation of microglia following ischemia remain unclear. In our experiment, we used digital gene expression (DGE) analysis to profile the transcriptome of ischemic tissue in a photothrombosis model. DGE analysis identified that a total of 749 genes were differentially regulated (643 up-regulated and 106 down-regulated) after 2days and 7days following stroke. We found 74.5% of these differentially expressed genes were microglial genes. Gene ontology (GO) analysis categorizes these differentially expressed genes at 2days and 7days to specific biological processes such as inflammatory response, cell activation, cell proliferation, and chemokine and cytokine production. Our data demonstrated that a large number of microglial genes were highly regulated at 2days after stroke, but the number of differentially expressed genes had reduced drastically by 7days. Importantly, some of the differentially expressed microglial genes at 7days did not show differential expression at 2days after stroke. DGE analysis indicated that specific genes related to microgliosis were regulated after ischemia. Consistent with the changes in transcriptome, the results from histological analysis of transgenic mice revealed that the microglia proliferated and aggregated surrounding the ischemic core during the period from 2days to 7days following photothrombosis. Together, these results suggested that transcriptomic changes in microglial genes after stroke may have a profound implication for pathophysiology and treatment of stroke.

Exogenous Neural Stem Cells Transplantation as a Potential Therapy for Photothrombotic Ischemia Stroke in Kunming Mice Model.

Stroke is considered as the second leading cause of death worldwide. The survivors of stroke experience different levels of impairment in brain function resulting in debilitating disabilities. Current therapies for stroke are primarily palliative and may be effective in only a small population of stroke patients. In this study, we explore the transplantation of exogenous neural stem cells (NSCs) as the potential therapy for the photothrombotic ischemia stroke in a Kunming mice model. After stroke, mice receiving NSC transplantation demonstrated a better recovery of brain function during the neurobehavioral tests. Histology analysis of the brain samples from NSC transplanted mice demonstrated a reduction of brain damage caused by stroke. Moreover, immunofluorescence assay for biomarkers in brain sections confirmed that transplanted NSCs indeed differentiated to neurons and astrocytes, consistent with the improved brain function after stroke. Taken together, our data suggested that exogenous NSC transplantation could be a promising therapy for stroke.

RfiA, a novel PAP2 domain-containing polytopic membrane protein that confers resistance to the FtsZ inhibitor PC190723.

BACKGROUND: As an essential protein for bacterial cell division, the tubulin-like FtsZ protein has been selected as a target for development of next generation antimicrobials. PC190723 is a fluoride-containing benzamide compound developed as a FtsZ inhibitor that selectively inhibits growth of multidrug resistant Gram-positive bacteria. AIM: Our aim was to investigate the mechanism of resistance to PC109723 conferred by over-expression of a gene, rfiA, in an environmental bacterium Arthrobacter A3. MATERIALS & METHODS: The investigations included analysis of the effect of PC109723 on wild-type Arthrobacter A3 and a recombinant strain over-expressing rfiA, in vivo localization of RfiA, in vitro measurements of fluorine release from PC109723 by membrane extracts from the over-expression strain combined with mass spectrophotometric analysis of reaction products, and modelling of RfiA structure. RESULTS: We describe a novel protein, RfiA, from Arthrobacter A3 that confers PC190723 resistance. RfiA is a PAP2 domain-containing polytopic transmembrane protein that can modify the fluoridated benzamide ring that is critical for high affinity binding of PC190723 with FtsZ. CONCLUSION: RfiA-mediated modification of PC190723 is the first reported instance of resistance to this antibiotic involving a change to its structure. We predict that adoption of PC190723 or related benzamides as antimicrobials in clinical practice will lead to the acquisition by resistant pathogens of a gene encoding this subfamily of proteins.

In vivo two-photon imaging of axonal dieback, blood flow, and calcium influx with methylprednisolone therapy after spinal cord injury.

Severe spinal cord injury (SCI) can cause neurological dysfunction and paralysis. However, the early dynamic changes of neurons and their surrounding environment after SCI are poorly understood. Although methylprednisolone (MP) is currently the standard therapeutic agent for treating SCI, its efficacy remains controversial. The purpose of this project was to investigate the early dynamic changes and MP's efficacy on axonal damage, blood flow, and calcium influx into axons in a mouse SCI model. YFP H-line and Thy1-GCaMP transgenic mice were used in this study. Two-photon microscopy was used for imaging of axonal dieback, blood flow, and calcium influx post-injury. We found that MP treatment attenuated progressive damage of axons, increased blood flow, and reduced calcium influx post-injury. Furthermore, microglia/macrophages accumulated in the lesion site after SCI and expressed the proinflammatory mediators iNOS, MCP-1 and IL-1ß. MP treatment markedly inhibited the accumulation of microglia/macrophages and reduced the expression of the proinflammatory mediators. MP treatment also improved the recovery of behavioral function post-injury. These findings suggest that MP exerts a neuroprotective effect on SCI treatment by attenuating progressive damage of axons, increasing blood flow, reducing calcium influx, and inhibiting the accumulation of microglia/macrophages after SCI.

Two-photon microscopy as a tool to investigate the therapeutic time window of methylprednisolone in a mouse spinal cord injury model.

PURPOSE: The aim of the present study was to explore the use of two-photon microscopy for investigating the therapeutic time window of methylprednisolone (MP) treatment after spinal cord injury (SCI). METHODS: Twenty-four YFP H-line mice were subjected to hemisection SCI and then divided into four groups. Group 1 received MP at 30 min post-injury; group 2 received MP at 8 h post-injury; group 3 received MP at 24 h post-injury; and group 4 received saline at 30 min post-injury. Post-injury axonal dieback was imaged in vivo using two-photon microscopy. After all imaging sessions, histological examination of the surviving neurons and microglial/macrophage accumulation was performed. RESULTS: Two-photon imaging revealed the degree of progressive axon damage after SCI. Group 1 exhibited a shorter axonal dieback distance and slower axonal dieback speed than groups 2, 3, and 4 (p < 0.01). MAP-2 staining revealed greater neuronal survival in group 1 than in groups 2, 3, and 4 (p < 0.05). F4/80 staining revealed greater microglial/macrophage density in groups 2, 3, and 4 than in group 1 (p< 0.05). CONCLUSIONS: MP therapy may help attenuate progressive axon damage, reduce neuronal death, and inhibit microglial/macrophage accumulation, especially when initiated shortly after SCI.